CN103074356B - Vector for knocking out streptomycete gene as well as constructing method and application of same - Google Patents
Vector for knocking out streptomycete gene as well as constructing method and application of same Download PDFInfo
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- CN103074356B CN103074356B CN201310023281.8A CN201310023281A CN103074356B CN 103074356 B CN103074356 B CN 103074356B CN 201310023281 A CN201310023281 A CN 201310023281A CN 103074356 B CN103074356 B CN 103074356B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a vector for knocking out a streptomycete gene as well as a constructing method and an application of the vector. A recombinant vector is a recombinant DNA (Deoxyribose Nucleic Acid) obtained by inserting a locus ClaI of pKC1139 into a glucanase gene. In actual application, a streptomycete target gene knockout vector can be obtained after a DNA fragment for knocking out a streptomycete target gene is inserted in a multiple clone site of the recombinant vector; after the streptomycete target gene knockout vector is introduced into streptomycete with the target gene to be knocked out, transformants with thiostrepton resistances are screened; cellulose degradation and screening are carried out on the transformants with the thiostrepton resistances; and specifically, a homologous double-exchange transformant can be obtained through inserting the transformants with the thiostrepton resistances into a cellulose screening flat plate and then carrying out Congo red staining on the transformants with the thiostrepton resistances so as to directly observe degradation situation of the cellulose. Therefore, the phenomenon of incomplete knockout of the gene due to false positive antibiotics resistance can be avoided. Meanwhile, PCR verifications of a large amount of transformants can be avoided. The vector for knocking out the streptomycete gene, disclosed by the invention, can be used for screening any gene knockout double-exchange transformant of strains with no glucanase yield.
Description
Technical field
The present invention relates to carrier and construction process and application for knocking out streptomyces gene.
Background technology
Streptomycete (streptomyces) is a kind of aerobic Gram-positive soil bacteria with branched mycelium structure, belongs to Prokaryota actinomycetales Streptomycetaceae, is one of microbe groups main in soil.Its G%+C% content is up to 70%-80%, is the highest class biology of the known G%+C% content of occurring in nature up to now.Although streptomycete belongs to prokaryotic organism, but but there is very complicated mechanism of cell differentiation, it is the good material of research gene expression and regulation mechanism in time, space and program, secondly streptomycete has abundant secondary metabolism diversity, the microbiotic of occurring in nature nearly 70% is produced by streptomycete and nearly edge actinomycetes thereof, in addition streptomycete also produces other useful secondary metabolites as antitumor drug, immunosuppressor, pest-resistant dose and the outer lytic enzyme of some other born of the same parents etc., is widely used in industry, agricultural and medical and health and life science field.Therefore, disclosing the research of streptomycete heredity, growth and metabolic regulation extremely payes attention to, but not clear for the research of the raw metabolic regulation mechanism of streptomycete at present, along with completing of the order-checking of streptomyces coelicolor, Avid kyowamycin and streptomyces griseus in recent years, more streptomyces gene group examining order is carried out, therefore the function of new gene and known is carried out to genetic analysis and compel to cut at enzyme, and the most important method of research gene is carried out gene knockout exactly.Gene knockout is recombinated by homology of chromosome exactly, replaces or interrupts certain gene, then observes its phenotype and changes.But the method for this gene knockout directly depends on the foundation of efficient gene engineered vector in the streptomycete of a certain genus or kind and genetic conversion system thereof, carry out the research of gene function to people can be screened more easily obtain gene knockout mutant strain.The current genetic conversion system about streptomyces, the genetic system that comprises gene knockout is set up substantially, substantially be all to carry the conjugal transfer of inducing from RP4 conjugal transfer initiation site oriT by carrying the method for plasmid conjugal transfer between belonging between intestinal bacteria and streptomycete of goal gene upstream and downstream homology arm and resistant gene, utilize on the tra gene that comprises in intestinal bacteria ET12567 helper plasmid pUZ8002 and shuttle vectors.Wherein the screening of double exchange transformant is all according to antibiotic resistance and susceptibility substantially, utilizes the technology of panel photocopy to engage that the methods such as pcr amplification, southern hybridization screen.But for replica screening time, there is false positive significantly due to antibiotics resistance and susceptibility; Can consume a large amount of human and material resources and directly screen by pcr amplification, because streptomycete is as gram positive bacterium, its cell walls is thicker, and the stability of bacterium colony PCR is very poor, so utilize the method bacterium of antibiotics resistance and pcr amplification can not improve the screening efficiency of double exchange transformant.
Summary of the invention
A technical problem to be solved by this invention be to provide using glucanase gene as selection markers gene for knocking out carrier and the construction process thereof of streptomyces gene.
Provided by the present invention for knocking out the carrier of streptomyces gene, name is called pKCG
+1139, be that glucanase gene is inserted to the recombinant DNA that the Cla I site of pKC1139 obtains.
Wherein, the physical map of pKC1139 as shown in Figure 1, pKC1139 plasmid belongs to streptomyces gene group damage type plasmid, can be used for gene disruption test, realize the checking to gene function, and can improve antibiotic output or product spore activity (Shen Fengying in some streptomycete by blocking some negative regulatory sequences, Li Yaning, Liu Liqiang, Wu Weigang, Liu great Qun. foundation and the optimization of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete Men-myco-93-63 genetic conversion system. Chinese agronomy circular 2009,25 (13): 197-201).
Following protein a) or b) of the concrete codified of above-mentioned glucanase gene:
A) protein of aminoacid sequence as shown in SEQ ID No.1;
B) by the replacement of one or several amino-acid residue in SEQ ID No.1 and/or disappearance and/or interpolation and have degraded cellulose activity by a) derivative protein.
Wherein, SEQ ID No.1 is made up of 499 amino-acid residues.
The encoding sequence of described glucanase gene specifically can be as shown in the 208-1707 position of SEQ ID No.2.
In one embodiment of the invention, the nucleotide sequence of described glucanase gene specifically can be the 9-1707 position of SEQ ID No.2.
Wherein, SEQ ID No.2 is made up of 1715 Nucleotide, and 1-8 position is Cla I recognition site and protection base, and 9-207 position is erythromycin promotor; the encoding sequence of 208-1707 position glucanase gene, 1708-1715 is Cla I recognition site and protection base.
Build the above-mentioned method for the carrier that knocks out streptomyces gene and also belong to protection scope of the present invention.
Contain the above-mentioned recombinant microorganism for the carrier that knocks out streptomyces gene and also belong to protection scope of the present invention.
Described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), streptomyces (Streptomyces) etc.
The above-mentioned gene that can be used for knocking out the streptomycete that does not produce dextranase for knocking out the carrier of streptomyces gene.
Wherein, the described streptomycete that does not produce dextranase can be streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.
In one embodiment of the invention, described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, described in knock out the streptomycete that does not produce dextranase gene for knocking out the synthetic positive regulating gene of tennecetin.
Another technical problem to be solved by this invention is to provide streptomycete goal gene knockout carrier.
Streptomycete goal gene knockout carrier provided by the present invention is to insert in the above-mentioned multiple clone site for the carrier that knocks out streptomyces gene the recombinant DNA obtaining for knocking out the DNA fragmentation of streptomycete goal gene.
Wherein, thus describedly substitute described goal gene by homologous recombination the goal gene of streptomycete knocked out for knocking out the DNA fragmentation of streptomycete goal gene.
The downstream homology arm of described upstream homology arm, thiostrepton resistant gene (tsr) and the described goal gene that can be followed successively by described goal gene for knocking out the DNA fragmentation of streptomycete goal gene from upstream to downstream.
Described streptomycete goal gene knockout carrier comprises thiostrepton resistant gene (tsr) and apramycin resistant gene (apm), does not therefore affect the screening of resistance transformant in intestinal bacteria and streptomycete in vector construction process.
Another technical problem to be solved by this invention is to provide a kind of method of preparing the streptomycete mutant strain that goal gene is knocked.
The method of preparing the streptomycete mutant strain that goal gene is knocked provided by the present invention, comprise the steps: above-mentioned streptomycete goal gene knockout carrier to import in the streptomycete of waiting to knock out described goal gene, obtain the transformant of thiostrepton resistance by thiostrepton resistance screening, from the transformant of described thiostrepton resistance, screen the not transformant of degraded cellulose, the transformant of the not degraded cellulose obtaining is the streptomycete mutant strain that candidate's goal gene is knocked;
In aforesaid method, described degraded cellulose is presented as degradation of sodium carboxymethylcellulo, e.
In aforesaid method, described in wait to knock out described goal gene streptomycete be the streptomycete that does not produce dextranase, as streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.
In one embodiment of the invention, described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, and described goal gene is the synthetic positive regulating gene slnM of tennecetin.The described nucleotide sequence for the DNA fragmentation that knocks out streptomycete goal gene is SEQ ID No.5.
The encoding sequence of the synthetic positive regulating gene slnM of described tennecetin is SEQ ID No.4, the protein of encoding amino acid sequence as shown in SEQ ID No.3.
Wherein, SEQ ID No.4 is made up of 579 Nucleotide, and SEQ ID No.3 is made up of 192 amino-acid residues.SEQ ID No.5 is made up of 4144 Nucleotide, 29-1221 position is the upstream homology arm of the synthetic positive regulating gene slnM of tennecetin, 1265-2911 position is thiostrepton resistant gene (tsr), the downstream homology arm of the synthetic positive regulating gene slnM of 2956-4117 position tennecetin.
Of the present invention for knocking out the carrier pKCG of streptomyces gene
+1139 will be placed under the erythromycin promotor that streptomycete is conventional from the glucanase gene of bacillus amyloliquefaciens, comprise the two negative selection markers apramycin resistant genes of gene knockout and glucanase gene.PKCG
+1139 can express glucanase gene in intestinal bacteria (as Escherichia coli BL21, E.coli DH5 α, E.coli Top10 etc.), streptomycete (as streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.), therefore can knock out negative selection markers as streptomyces gene and use.In actual applications, can be at pKCG
+1139 multiple clone site obtains streptomycete goal gene knockout carrier after inserting the DNA fragmentation for knocking out streptomycete goal gene, this streptomycete goal gene knockout carrier is imported to the transformant that screens thiostrepton resistance after knocking out the streptomycete of described goal gene, the transformant of thiostrepton resistance is carried out to cellulose degradation screening again, specifically can obtain homology double exchange transformant by carrying out again the cellulosic degraded situation of the direct observation of congo red staining on the transformant incoming fiber element screening flat board of thiostrepton resistance.The phenomenon that does not knock out gene completely that can avoid like this antibiotics resistance false positive to cause.Can avoid a large amount of transformant PCR checkings simultaneously.Can be used in the screening of any gene knockout double exchange transformant that does not produce dextranase bacterial strain.
Of the present invention for knocking out the carrier pKCG of streptomyces gene
+1139 have overcome the deficiency that current gene knockout carrier occurs false positive and need to expend a large amount of human and material resources, are a kind of gene knockout carriers with selection markers directly perceived, can carry out streptomycete chromosome gene fast, easily and knock out.This selection markers gene directly perceived is glucanase gene, can directly observe and obtain gene knockout double exchange transformant by the method for congo red staining, make up the false-positive shortcoming due to antibiotics resistance, for the screening of transgenation strain provides more practical instrument.
By pKCG
+m
-1139 carriers transform demethylation E.coli ET12567 (pUZ8002), the method engaging by amphiphilic, transform streptomyces lydicus, by thiostrepton resistance screening, 500 strains of picking positive transformant, and went down to posterity for 5 generations in thiostrepton resistant panel, carry out Xylo-Mucine plate screening, congo red staining.Result shows 496 strain transformants and shows dextranase activity, there are 4 strains there is no dextranase activity, whether the bacterial strain without dextranase activity is carried out to pcr amplification checking goal gene knocks out, result shows that 4 strains do not produce the bacterial strain of dextranase and all do not increase and obtain goal gene, and obtain the fragment of 1.0kb of expection, and produce the bacterial strain PCR result of the dextranase goal gene fragment that all increased, therefore utilize this carrier one step can obtain intuitively double exchange mutant strain, saved a large amount of manpower and materials.In order further to verify the reliability of the method, to after remaining 496 strain transformant shaking flask 5 generations of subculture, carry out dextranase biopsy survey, filter out the bacterial strain that 3 strains do not have dextranase activity, the stepping performing PCR of going forward side by side is verified, result shows, all dextranase negative strains all do not increase and obtain goal gene, therefore illustrate that the method is accurate, easy, can be applied to the screening operation of double exchange mutant strain.
Brief description of the drawings
Fig. 1 is the physical map of pKC1139.
Fig. 2 is pKCG
+1139 physical map.
Fig. 3 is pKCG
+m
-1139 physical map.
Fig. 4 is the streptomycete mutant strain that is knocked of slnM and the PCR electrophoretogram of wild strain.
Wherein, swimming lane M is DNA molecular amount standard, and A02 is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, A02-slnM
-streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain being knocked for slnM.
Fig. 5 is the bacterium colony photo of congo red staining on Mierocrystalline cellulose culture medium flat plate.
Arrow shows streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain (A02-slnM that slnM is knocked
-).
Fig. 6 A is that the HPLC of Streptomyces lydicus A02 CGMCC No.1654 active result separates spectrogram for the first time.
Fig. 6 B is that the HPLC of Streptomyces lydicus A02 CGMCC No.1654 active result separates spectrogram for the third time.
Fig. 7 is the UV scanning collection of illustrative plates of tennecetin sample.
Fig. 8 is the infrared absorption spectrum of tennecetin sample.
Fig. 9 A is the high resolution mass spectrum figure (negative ion) of tennecetin sample.
Fig. 9 B is the high resolution mass spectrum figure (positive ion) of tennecetin sample.
Figure 10 A is the carbon-13 nmr spectra (500MHz) of tennecetin sample.
Figure 10 B is the proton nmr spectra (500MHz) of tennecetin sample.
Figure 11 is the bacterium colony photo of congo red staining on the Mierocrystalline cellulose culture medium flat plate of comparative example.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Streptomyces expression vector pIB139(Christopher J.Wilkinson in following embodiment, et al.Increasing the Efficiency of Heterologous Promoters in Actinomycetes.J Mol Microbiol Bioteh (2002) 4 (4): 417 – 426), pKC1139(Shen Feng English, Li Yaning, Liu Liqiang, Wu Weigang, Liu great Qun. foundation and the optimization of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete Men-myco-93-63 genetic conversion system. Chinese agronomy circular 2009, 25 (13): the 197-201 public can obtain from Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
Demethylation E.coliET12567 (pUZ8002) (Sun-Uk Choi in following embodiment, Chang-Kwon Lee, Yong-Il Hwang, Hiroshi Kinoshita, and Takuya Nihira (2004) Cloning and Functional Analysis by Gene Disruption of a Gene Encoding a γ-Butyrolactone Autoregulator Receptor from Kitasatospora setae.JOURNAL OF BACTERIOLOGY, 186:3423 – 3430.) public can obtain from Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
Embodiment 1, structure are for knocking out the knockout carrier of the streptomyces gene that does not produce dextranase
1, the clone of bacillus amyloliquefaciens glucanase gene
It is template that the strain obtaining taking this laboratory screening has remarkable inhibiting bacillus amyloliquefaciens YW26 genomic dna to various plants pathogenic fungi, according to bacillus amyloliquefaciens endo glucanase gene (DQ782954.1) primers: P1(5'-ATGAAACGGTCAATTTCTAT-3 ' in GenBank) and P2(5'-CTAATTGGGTTCTGATCC-3'), carry out pcr amplification.Reaction is at 95 DEG C of denaturation 5min; 30 circulations (94 DEG C of 40s, 55 DEG C of 30s, 72 DEG C of 1min); 72 DEG C are extended 10min.The fragment of the 1500bp size that amplification is obtained is connected with plasmid pMD18-T carrier, obtains pMD18-glu, and conversion bacillus coli DH 5 alpha, carries out PCR by ammonia benzyl resistance screening by the positive transformant obtaining and enzyme is cut checking.Utilize universal primer order-checking, GenBank database BLAST analyzes inscribe β-1 that shows this sequence and a strain Bacillus subtilis strain C-36,4-glucanase gene sequence identity reaches 100%, thereby determines that this gene is glucanase coding gene as well as.The nucleotide sequence of the glucanase coding gene as well as PCR product that in pMD18-glu, P1 and P2 obtain is the 208-1707 position of SEQ ID No.2, the dextranase of coding SEQ ID No.1.
2, the structure of streptomyces expression vector
The promotor of utilizing the erythromycin promotor (ermE*) of carrying on streptomyces expression vector pIB139 to express as glucanase gene, builds and obtains streptomycete dextranase expression vector pIB139-glu.Concrete grammar is as follows:
According to the analysis to pIB139 carrier restriction enzyme site, suitable restriction enzyme site NdeI and EcoRI are added to F15'-in glucanase gene primer
gGAATTCCATATGand R15'-ATGAAACGGTCAATCTCTAT-3'(NdeI)
cGGAATTCcTAATTTGGTTCTGTTC-3'(EcoRI), obtain the dextranase with restriction enzyme site taking pMD18-glu as template amplification, by NdeI/EcoRI double digestion and cut through same enzyme the streptomyces expression vector pIB139 that obtains carrying erythromycin promotor (ermE*) and be connected, obtain pIB139-glu, and transform bacillus coli DH 5 alpha, with apramycin (apramycin) thus resistance screening obtains takes the transformant of glucanase gene, obtain the fragment of about 1.5kb by pcr amplification, enzyme is cut the glucanase gene fragment of verifying the pIB139 plasmid and the about 1.5kb that obtain 5.9kb, thereby determine that fragment exact connect ion is in pIB139 carrier.
3, clone obtains the glucanase gene under erythromycin promoters driven, and builds the streptomyces gene knockout carrier that carries dextranase selection markers
With erythromycin promoter sequence upstream primer p3(
cCATCGATand under glucanase gene, have a promotor p4(CTAGTATGCATGCGAGTG)
cCATCGATcTAATTTGGTTCTGTTC) (line part is restriction enzyme ClaI site and protection base) is primer; taking the streptomyces expression vector pIB139-glu that carries erythromycin promotor (ermE*) as template; amplification obtains the fragment ermE-glu that nucleotide sequence is erythromycin promotor and the glucanase gene sequence of SEQ ID No.2; this fragment is inserted after ClaI enzyme is cut to the ClaI site of pKC1139, obtained the recombinant expression vector pKCG of erythromycin promotor and glucanase gene insertion pKC1139
+1139(Fig. 2).PKCG
+1139 be the present invention protection for knocking out the knockout carrier of the streptomyces gene that does not produce dextranase.
Embodiment 2, structure knock out the knockout carrier of the synthetic positive regulating gene slnM of tennecetin in the streptomycete that does not produce dextranase
The encoding sequence of the synthetic positive regulating gene slnM of tennecetin is SEQ ID No.4, the protein of encoding amino acid sequence as shown in SEQ ID No.3.
First building nucleotide sequence is the DNA fragmentation that knocks out streptomycete slnM of SEQ ID No.5; SEQ ID No.5 is made up of 4144 Nucleotide; 1-8 position is EcoRI recognition site and protection base; 29-1221 position is the upstream homology arm of the synthetic positive regulating gene slnM of tennecetin; 1265-2911 position is thiostrepton resistant gene (tsr); the downstream homology arm of the synthetic positive regulating gene slnM of 2956-4117 position tennecetin, 4136-4144 position is HindIII recognition site and protection base.
The DNA fragmentation that knocks out streptomycete slnM that by nucleotide sequence is SEQ ID No.5 inserts pKCG after Hind III and EcoR I enzyme are cut
+1139 Hind III and EcoR I site obtain slnM knockout carrier pKCG
+m
-1139(Fig. 3).
Embodiment 3, build and do not produce the mutant strain that in the streptomycete of dextranase, the synthetic positive regulating gene slnM of tennecetin is knocked
One, by pKCG
+m
-1139 carriers transform demethylation E.coliET12567 (pUZ8002)
By heat shock method by pKCG
+m
-1139 carriers import E.coliET12567 (pUZ8002) and obtain proceeding to pKCG through 15 μ g/ml thiostrepton resistance screenings
+m
-recombinant bacterium E.coliET12567 (the pUZ8002)/pKCG of 1139 carriers
+m
-1139.
Two, slnM knocks out the preparation of strain
Reference (Bierman M et al.1992) carries out amphiphilic joint by pKCG
+m
-1139 import streptomyces lydicus, and concrete grammar is as follows:
By recombinant bacterium E.coliET12567 (pUZ8002)/pKCG
+m
-1139 are inoculated into the LB liquid nutrient medium that contains paraxin, kantlex and apramycin resistance, and 37 DEG C, 200rpm, overnight incubation, receive in fresh LB substratum and be cultured to OD morning next day by 1% inoculum size
600=0.4-0.6, collects bacterium liquid, and 4 DEG C, 4000rpm, centrifugal 2min, abandons supernatant, by the ice-cold pure resuspended precipitation of LB nutrient solution of 20mL, 4 DEG C, 4000rpm, centrifugal 2min, abandons supernatant, and object is to wash away microbiotic.By the ice-cold pure resuspended precipitation of LB nutrient solution of 2mL.(2) streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654(CN100467588C being grown 2 weeks in PDA inclined-plane) spore aseptic double-distilled water elutes, and with sample injector piping and druming evenly, prepare spore suspension.(3) 250 μ L streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 spore suspensions add 500 μ L2 × YT nutrient solutions, mix gently.50 DEG C of thermal shock 10min, activation spore.500 μ L recombinant bacterium E.coliET12567 (pUZ8002)/pKCG
+m
-streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 spore suspension that 1139 bacterium liquid and 500 μ L activate, mixes gently.4000rpm, the centrifugal 3min of room temperature, removes supernatant, mixes precipitation and coats MS substratum+10mM/L MgCl
2, 28 DEG C of inversions are cultured to morning next day (18h), are coated with afterwards antibiotic solution and (0.5mg nalidixic acid and 60 μ g apramycins are dissolved in to 1mLddH
2the liquid obtaining in O).After 2-3 days, picking list bacterium colony is to the new MS flat board that adds above-mentioned antibiotic solution.The bacterium colony of picking on this MS flat board adds on MS+15 μ g/ml thiostrepton flat board (adding thiostrepton to make its final concentration in MS is 15 μ g/ml), screening obtains transformant 500 strains of thiostrepton resistance, 5 generations of continuous passage, transformant is inoculated on Mierocrystalline cellulose culture medium flat plate and is cultivated 2 days at 29 DEG C, carry out congo red staining, showing 496 strain transformants by congo red staining and produce obvious transparent circle, have the ability of degraded cellulose, is degraded cellulose transformant, there are 4 strains not produce transparent circle, for degraded cellulose transformant not, by not bacterial strain primer (the forward primer atggcgagccttgataaaac of degraded cellulose of this four strain, reverse primer tcacttcacgaagtcgtcc) whether pcr amplification checking goal gene slnM be knocked, result show 4 strains not the bacterial strain of degraded cellulose all do not increase and obtain goal gene, and obtain the fragment (Fig. 4) of 180bp of expection, and wild type strain streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654PCR amplification obtains the fragment of 579bp, show 4 plant mutant strain generation double exchanges, pKCG
+m
-carrier after 1139 carrier generation double exchanges from streptomyces lydicus (Streptomyces lydicus) thus A02CGMCC No.1654, disappear and obtain streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain (Fig. 5) that is knocked of slnM, and produce the bacterial strain PCR result of the dextranase goal gene fragment (fragment of 579bp) that all increased, illustrate that single cross has only occurred changes.Therefore utilize this carrier one step can obtain intuitively double exchange mutant strain, saved a large amount of manpower and materials.In order further to verify the reliability of the method, the detection of cellulose degradation ability will be carried out after remaining 496 strain transformant shaking flask 5 generations of subculture according to the method described above, filter out the not bacterial strain of degraded cellulose of 2 strains, and further use primer (forward primer atggcgagccttgataaaac, reverse primer tcacttcacgaagtcgtcc) whether pcr amplification checking goal gene slnM be knocked, result shows, this 2 strain does not have the ability of degraded cellulose, all do not increase and obtain goal gene, and obtain the fragment of 180bp of expection, therefore illustrate that the method is accurate, easy, can be applied to the screening operation of double exchange mutant strain.
In addition, streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is inoculated on Mierocrystalline cellulose culture medium flat plate and is cultivated 2 days at 29 DEG C, carry out congo red staining, result shows that wild-type streptomycete bacterial strain streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 does not produce transparent circle, not degraded cellulose, does not have dextranase activity.
MS substratum in the present embodiment:
Mierocrystalline cellulose substratum in the present embodiment: Xylo-Mucine 20g, Na
2hPO
42.5g, KH
2pO
42.5g, peptone 2.5g, yeast extract paste 0.5g, be settled to 1000mL with distilled water.
Three, streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that slnM is knocked and the comparison of wild strain tennecetin synthesis capability
In this embodiment, substratum used is as follows:
Gause I slant medium: K
2hPO
40.5g, NaCl0.5g, KNO
31.0g, FeSO
47H
2o0.01g, MgSO
47H
2o0.5g, Zulkovsky starch 20g, agar 20g, water 1000ml; PH7.2-7.4.
Seed culture medium: 15g soybean grain (adding distil water boils 0.5-1h, gets filtrate), 5g peptone, 2.5g ammonium sulfate, 20g glucose, 10g starch, 0.25g magnesium sulfate, 0.2g potassium primary phosphate, 5g sodium-chlor, be made into the aqueous solution, adjust after pH7-8, add 1g calcium carbonate, add water and be settled to 1000ml.
Fermention medium: 15g soybean grain (adding distil water boils 0.5-1h, gets filtrate), 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g magnesium sulfate, 0.2g potassium primary phosphate, 5g sodium-chlor, be made into the aqueous solution, adjust after pH7-8, add 1g calcium carbonate, add water and be settled to 1000ml.
1, the preparation of streptomyces lydicus (Streptomyces lydicus) natamycin fermentation liquor
Streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that the slnM that streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 and step 2 are obtained is knocked is inoculated into respectively on Gause I slant medium, cultivate 7-10 days for 28 DEG C, treat that it produces enough spores, encircle with aseptic its spore of platinum loop scraping 2-3 in the 50ml seed culture medium being inoculated in 250ml triangular flask, put on temperature controllable shaking table, under 28 DEG C of conditions, 200rpm(rotation radius 13mm) constant-temperature shaking culture 24h-30h; Then its point is connected to (every bottled liquid measure is 100ml) in 10 fermention mediums in 500ml triangular flask under aseptic condition, the inoculum size of streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that the slnM that streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 obtains with step 2 is knocked is identical, OD after every bottle graft kind
600value is 0.1; Postvaccinal shaking flask is under 31 DEG C of conditions, with 240rpm(rotation radius 13mm) speed oscillation cultivate 96h.
2, tennecetin volume analysis
The 1mL fermented liquid that step 1 is obtained adds 9mL methyl alcohol, fully after vibration, carry out 30min ultrasonic extraction, 5000rpm/min centrifugal 10-15min sedimentation mycelium and solid substance, supernatant liquor dilutes 10 times of aseptic filtering with microporous membrane of using afterwards 0.45 μ m, collect filtrate, gained sample detects for HPLC.Chromatographic column: C18 post (5 μ m, 4.6mm × 200mm); Detect wavelength: 303nm; Flow velocity: 1.00mL/min; Sample size: 10 μ L; Detect column temperature: 30 DEG C; Experiment moving phase is V (methyl alcohol): V (water)=65: 35.Adopt external standard method (calibration curve method) to carry out quantitatively tennecetin taking tennecetin (sigma-P9703) as standard substance.
Experimental result shows to can't detect tennecetin in the fermented liquid of streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that slnM that step 2 obtains is knocked, and the tennecetin output of streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is 2.546g/L.In streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that the slnM that description of step two obtains is knocked, positive regulating gene slnM is successfully knocked, make transgenation strain not there is the ability of synthetic tennecetin, in this Natal streptomycete of reporting with Ant ó n et al.2007, knocking out of approach specificity positive regulating gene pinM gene (homologous gene of Streptomyces lydicus A02 slnM gene) causes that the forfeiture of tennecetin synthesis capability is consistent, this experimental result further shows that dextranase is as the negative selection markers of gene knockout, fast, easy, save time, laborsaving, can be widely used in streptomycete and other gene knockout mutant strain seed selection work.
Wherein, in Streptomyces lydicus A02 CGMCC No.1654 fermented liquid tennecetin slightly carry with authentication method as follows:
One) slightly the carrying of tennecetin in fermented liquid
The fermented liquid of above-mentioned streptomyces lydicus is used respectively to the dehydrated alcohol pre-treatment of 3 times of volumes, 4 DEG C of standing 2h, with precipitation thalline, solid particles, solubility sticky jelly, nucleic acid and heteroproteins and mesostate etc., supernatant liquor with 2 metafiltration paper with Büchner funnel vacuum filtration, filtrate is through Rotary Evaporators concentrating under reduced pressure at 45 DEG C, concentrated solution is tennecetin crude extract, and 4 DEG C save backup.
Two), the separation and purification of tennecetin crude extract
Carry out progressively separation and purification by macroporous resin column chromatography, silica gel column chromatography and high performance liquid chromatograph HPLC.
1, macroporous resin adsorption column chromatography
Select 40cm × 2.6cm glass chromatography column, X-5 macroporous resin (Tianjin Nankai university chemical plant).Macroporous resin is mixed well by appropriate amount of deionized water after pre-treatment being described by producer, slowly adds in the chromatography column that 1/3 volumes of deionized water is housed, and emits distilled water from column bottom with uniform speed slow simultaneously, and liquid level in post is remained at above resin layer.Be filled to approximately 3/4 post height, natural subsidence 6～10h, making the dress column volume after balance is 150ml.
Get tennecetin crude extract and resin and carry out dynamic adsorption by the volume ratio of 1 ﹕ 1.Elution process is: the deionized water wash-out remove portion pigment of 2 times of column volumes and a large amount of water-soluble impurity; The 30%(volumn concentration of 2 times of column volumes) the methanol-eluted fractions element that discolors; Finally use the 70%(volumn concentration of 2 times of column volumes) ethanol elution activeconstituents.Be in charge of collection elutriant, every pipe 15ml, filter paper method is carried out determination of activity.
Result shows that active eluant concentrates on 48th～56 pipes (i.e. elutriant between 4.8 times of column volume to 5.6 times column volumes).By active eluant, at 45 DEG C after concentrating under reduced pressure for next step separation.
2, silica gel adsorption column chromatography
Select 40cm × 2.6cm glass chromatography column, 100 order～200 order silica gel, the mixed solution taking volume ratio as ethanol, ammoniacal liquor and the water of 8 ﹕ 1 ﹕ 1 is as elutriant; Get about 150g silica gel deionized water and soak 3h, the fine particle that inclines, Büchner funnel vacuum filtration is removed moisture; Soak 12h with 6mol/L HCl again, be then washed till neutrality with deionized water, vacuum is drained; Spend the night by soaked in absolute ethyl alcohol, vacuum is drained; Under 120 ° of C, activate 2h before use, be dried to constant weight; In chromatography column, pack the elutriant of 1/3 column volume into, then slowly add the silica gel mixing with elutriant, approximately to 3/4 post stops adding when high, leaves standstill 6～10h, makes the slow sedimentation of silica gel.Then rinse cylinder with the elutriant of 2～3 times of volumes with the flow velocity of 1mL/min, make it balance.Column volume after balance is 150ml.The active wash-out concentrated solution of the macroporous resin 10ml upper prop of getting step 1 carries out dynamic adsorption, carries out wash-out with the elutriant of 2～3 times of column volumes by the flow velocity of 0.5ml/min, is in charge of collection elutriant, every pipe 5ml with automatic Fraction Collector.Taking S. cervisiae ACCC20036 as indicator, utilize filter paper agar diffusion method to detect every pipe elutriant activity.Result shows: the active ingredient in its elutriant concentrates on 7th～36 pipes (i.e. elutriant between 0.23 times of column volume to 1.2 times column volume).By active eluant, at 45 DEG C after concentrating under reduced pressure for next step separation.
3, preparation HPLC separation and purification
Adopt LC-9101 type circulation preparative HPLC, JAIGEL-ODS-AP type SP-120-15 preparative column.
Get the active wash-out concentrated solution after the silica gel column chromatography of step 2, filter automatic sampler sample introduction, each sample size 6ml with 0.45 μ m millipore filter; Taking methyl alcohol: water (volume ratio is as 7:3), as moving phase separates, utilizes UV detector to detect at wavelength 305nm place and automatically forms separating spectrum; Utilize run tank to collect respectively the corresponding elutriant in each curve peak in collection of illustrative plates; Taking S. cervisiae ACCC20036 as indicator, utilize filter paper agar diffusion method to detect every pipe elutriant activity.After separating for the first time with the flow rate pump of 2ml/min, more in succession separate 2 times with the flow rate pump of 3ml/min.
Experimental result shows, HPLC separates and 30 peaks detected altogether for the first time, and the strong absorption peak that wherein retention time is 57.866min is Peak Activity (Fig. 6 A), and its relative peak area is 35.121%; The peak of separation detection to 6 for the second time that it is carried out, the peak that wherein retention time is 41.699min is Peak Activity, its relative peak area is 97.020%; The peak that separation detection is 39.766min to retention time is for the third time Peak Activity (Fig. 6 B), is 99.845% by its purity of calculated by peak area.
Carry out vacuum concentration by separating for the third time the Peak Activity sample obtaining, after being dried, be white in color or cream-colored powder, this sample is tennecetin sample.Utilize Shimadzu analysis mode HPLC to adopt following method to carry out purity checking to it: the sample that takes a morsel is dissolved in 70% methanol aqueous solution, carry out gradient elution taking methyl alcohol (A) and water (B) as moving phase.Chromatographic condition is: C
18reversed-phase column, 30 DEG C of column temperatures, UV detector, detects wavelength 305nm, and SIL-10ADVP automatic sampler sample introduction 10 μ l, with the flow velocity wash-out 60min of 1ml/min.Gradient elution step is as follows:
Result shows that elution curve is single peak, illustrates that it is one-component, and purity reaches the requirement of determination of chemical structure.
Four, the parsing of purification of samples chemical structure qualification
1, ultra-violet absorption spectrum (UV)
The tennecetin sample denier of above-mentioned steps three purifying is dissolved in ultrapure water, use the UV of Hitachi-VIS3010 ultraviolet-visible pectrophotometer carries out full wavelength scanner in 190nm～400nm wavelength region taking ultrapure water as blank, automatically form ultraviolet absorpting spectrum.
From UV scanning collection of illustrative plates, tennecetin sample shows typical tetraenes microbiotic spectral pattern, near wavelength 281nm, 291nm, 305nm and 319nm, all there is the absorption peak of typical conjugation tetraene chromophoric group, its medium wavelength 305nm place absorption value maximum, 281nm place absorption value minimum (Fig. 7), illustrates that this material belongs to the tetraene microbiotic in polyenoid class.
2, infrared absorption spectrum (IR)
Adopt KBr pressed disc method, carry out 400cm with German BRUKER company's T ENSOR27 Fourier infrared spectrograph
-1-4000cm
-1region interscan.
The infrared spectra of tennecetin sample as shown in Figure 8, wherein ν
max3416.78cm
-1for the charateristic avsorption band of-OH; ν
max3288.23cm
-1for the stretching vibration charateristic avsorption band of N-H; ν
max2940.44 and 2980.27cm
-1be-CH
3charateristic avsorption band; ν
max3017.23cm
-1be-CH
2charateristic avsorption band; ν
max1715.38cm
-1show the strong absorption peak of typical carbonyl; ν
max1571.44cm
-1the strong absorption peak of performance-C=C-; ν
max1634.40cm
-1the weak absorption peak of performance-C=C-.
3, high resolution mass spectrum
Adopt the German BRUKER ultrahigh resolution 9.4T of company mixed type level Four bar fourier tandom mass spectrometer (9.4T Q-FT-MS); Condition: capillary4000, Dry Gas:4.0l/s, source temperature: 180 ° of C, scan range:300～2000, syringe pump:1.5ml/min, data analysis software is Bruker Daltonics DataAnalysis3.4.
Result shows, in the collection of illustrative plates that tennecetin sample is analyzed, adopts positive ion detection mode adduct ion [M+Na] to be detected
+for m/z688.2937(Fig. 9 B); Adopt negative ion detection mode quasi-molecular ion [M-H] to be detected
+for the compound (Fig. 9 A) of m/z664.2975; Adopt positive and negative ion detection mode to detect analysis to tennecetin sample, determine that 664.2975 for molecular ion peak.
In sum, the molecular formula of the main active component of tennecetin sample is C
33h
47nO
13, molecular weight is 665; By formula: degree of unsaturation (n)=1+Nc+(Nn-Nh)/2(Nc: carbonatoms; Nn: nitrogen-atoms number; Nh: number of hydrogen atoms) degree of unsaturation of calculating its molecular formula is 11, shows to contain in its molecular structure multiple unsaturated link(age)s and ring etc.
4, nuclear magnetic resonance spectrum (NMR)
Taking deuterated-dimethyl formamide (d-DMF) as solvent, taking tetramethylsilane (TMS) as interior mark, under room temperature, measure.
Adopt Bruker AVANCE DRX-500 nuclear magnetic resonance spectrometer (German Bruker spectral instrument company), taking deuterated-dimethyl formamide (d-DMF) as solvent, tetramethylsilane (TMS) is interior mark, carry out hydrogen spectrum (
1hNMR) and carbon spectrum (
13cNMR) mensuration; The former resonant frequency is 500.1325156MH
z, sampling number 32768 times; The latter's resonant frequency 125.7577612MH
z, sampling number is 65536 times.
Experimental result shows, the nucleus magnetic resonance of tennecetin sample
13in C collection of illustrative plates (Figure 10 A), can find out in molecule, there is the chemical shift (δ 165.217) of a carboxyl carbon atom; The chemical shift (δ 178.603) of a carbonylic carbon atom; The chemical shift (δ 125.089～145.447) of one group of conjugation tetraene carbon atom; The chemical shift (δ 66.102～70.326) of one group of carbon atom being connected with hydroxyl; Sugar upper five the carbon atom resonance peaks of ring (δ 71.194～97.894); Methine carbon atom resonance peak (δ 18.062, δ 20.360).The nucleus magnetic resonance of 500 megahertzes
1in H collection of illustrative plates (Figure 10 B), can find out the chemical shift (δ 5.686～6.625ppm) of the proton hydrogen (CH=CH-) being connected with four two keys on polyenoid ring; The chemical displacement value (δ 4.187～4.741ppm) of the proton hydrogen in five hydroxyls; The chemical shift (δ 1.274～2.439ppm) of the proton hydrogen in five methylene radical and two methyl.
The above-mentioned experimental data of comprehensive analysis, the main active component of tennecetin sample is tennecetin, its chemical structural formula is:
Comparative example, glucanase gene under erythromycin promoters driven is inserted to the ClaI of pKC1139 and the recombinant vectors that KpnI site obtains is not expressed glucanase gene
With erythromycin promoter sequence upstream primer p3(
cCATCGATcTAGTATGCATGCGAGTG) under (line part is restriction enzyme ClaI site and protection base) and glucanase gene, there is promotor p4(
gGGTACCgGATCAGAACCCAATTAG) (line part is restriction enzyme KpnI site and protection base) is primer; taking the streptomyces expression vector pIB139-glu that carries erythromycin promotor (ermE*) as template; amplification obtains the fragment ermE-glu of erythromycin promotor and glucanase gene sequence; this fragment is inserted between the ClaI and KpnI site of pKC1139 after ClaI and KpnI enzyme are cut, obtained erythromycin promotor and glucanase gene and insert the recombinant expression vector pKCG1139-2 of pKC1139.
By heat shock method by the pKCG of embodiment 1
+1139 and pKCG1139-2 transform respectively bacillus coli DH 5 alpha and through 100 μ g/ml apramycin resistance screenings, obtain carrying carrier pKCG
+bacillus coli DH 5 alpha transformant (called after DH5 α-pKCG of 1139
+1139) and carry the bacillus coli DH 5 alpha transformant (called after DH5 α-pKCG1139-2) of pKCG1139-2.By DH5 α-pKCG
+1139 and DH5 α-pKCG1139-2 be inoculated in respectively on the Mierocrystalline cellulose culture medium flat plate of embodiment 3 and cultivate 2 days at 29 DEG C, carry out congo red staining, result shows that DH5 α-pKCG1139-2 does not produce transparent circle, not degraded cellulose, does not have dextranase activity; DH5 α-pKCG
+1139 produce transparent circle, have dextranase activity.The recombinant vectors pKCG that ClaI site single endonuclease digestion obtains is described
+1139 express glucanase gene, and the recombinant vectors pKCG1139-2 that ClaI and KpnI site obtain does not express glucanase gene (Figure 11), show that ClaI and KpnI double digestion may cut away the essential site on pKC1139 carrier, cause that dextranase can not express.
Claims (7)
1. the method that builds recombinant vectors, is the Cla I site of glucanase gene being inserted to pKC1139, and the recombinant DNA obtaining is described recombinant vectors; The nucleotide sequence of described glucanase gene is the 9-1707 position of SEQ ID No.2.
2. the recombinant vectors being built by method described in claim 1.
3. contain the recombinant microorganism of recombinant vectors described in claim 2.
4. recombinant vectors claimed in claim 2 is in the application knocking out in the streptomyces gene that does not produce dextranase.
5. application according to claim 4, is characterized in that: the described streptomycete that does not produce dextranase is streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete or brown yellow spore streptomycete.
6. streptomycete goal gene knockout carrier is to insert in the multiple clone site of recombinant vectors claimed in claim 2 the recombinant DNA obtaining for knocking out the DNA fragmentation of streptomycete goal gene.
7. prepare the method for the streptomycete mutant strain that goal gene is knocked for one kind, comprise the steps: streptomycete goal gene knockout carrier claimed in claim 6 to import in the streptomycete of waiting to knock out described goal gene, obtain the transformant of thiostrepton resistance by thiostrepton resistance screening, from the transformant of described thiostrepton resistance, screen the not transformant of degraded cellulose, the transformant of the not degraded cellulose obtaining is the streptomycete mutant strain that candidate's goal gene is knocked; Described streptomycete of waiting to knock out described goal gene is the streptomycete that does not produce dextranase.
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